This study reports the purification and characterization of β-glucosidase from a newly isolated thermophilic fungus, Melanocarpus sp. Microbial Type Culture Collection (MTCC) 3922. The molecular weight of βglucosidase was determined to be ~ 92 and 102 kDa with SDS PAGE and gel filtration, respectively, and pI of ~ 4.1. It was optimally active at 60ºC and pH 6.0, though was stable at 50ºC and pH 5.0-6.0. The presence of *Corresponding author DTT, mercaptoethanol and metal ions such as Na + , K + , Ca 2+ , Mg 2+ and Zn 2+ positively influenced the activity of β-glucosidase but the activity was inhibited in the presence of CuSO 4. β-Glucosidase recognized pNP-βglucopyranoside (pNPG) as the preferred substrate, and showed very low affinity for pNP-β-D-cellobioside. K m and V max for the hydrolysis of pNPG by β-glucosidase was calculated as 3.3 mM and 43.68 µmolmin-1 mg
Sarkar et al.: Reverse Phase High Performance Liquid Chromatography Method for Estimation of Piperine andCoenzyme Q10Today people are focused on their health, as good health is a key to the happiness of our life. Medicine takes a big responsibility to take care of our health. Various supplements are available to meet the nutritional requirement. One such supplement is a combination of piperine and coenzyme Q10. The medicines have to be analyzed properly before introducing them to the market. As no analytical method is available for simultaneous estimation of the combination, hence an attempt was made to develop a validated method for the estimation of piperine and coenzyme Q10 simultaneously in bulk and its dosage form. A simple, precise, accurate and validated reversed phase high performance liquid chromatography technique was developed for the estimation of piperine and coenzyme Q10 in bulk and its tablet formulation. In this developed method, Waters X Bridge C 18 column (250 mm×4.6 mm, 5 µm) was used as a stationary phase and acetonitrile, tetrahydrofuran and water was used in 65:32:3 (v/v) ratio as mobile phase with 1 ml/min flow rate. This isocratic separation was accomplished using Waters 2707 Autosampler high performance liquid chromatography system, Waters 515 solvent delivery system, with photodiode array detector detection at 275 nm. Chromatographic data was processed by Empower 2 software. The retention times of coenzyme Q10 and piperine were 4.56 and 8.19 min respectively. The linearity ranges have lied between 4-6 µg/ml, 240-360 µg/ml for piperine and coenzyme Q10 respectively with 0.997 as correlation coefficient for both.
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